Amides as inhibitors of human secreted phospholipase A2

ABSTRACT

Methods and compounds useful for inhibiting a phoshpolipase A 2  are provided, the methods comprising contacting the phoshpolipase A 2  with a compound having the structure A, or pharmaceutically acceptable salts thereof: wherein R 1  is H, F, NH 2 , or COOH; R 2  is, H, linear saturated or unsaturated alkyl, alkenyl, or alkynyl; each of R 3  and R 4  is independently H, linear saturated or unsaturated alkyl, alkenyl, alkynyl, phenyl, or substituted phenyl; R 5  is H, (C 1 -C 6 ) alkyl such as methyl or ethyl; X is aryl or substituted aryl, such as phenyl or a substituted phenyl; and Y is O or S.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 USC §371 National Stage application ofInternational Application No. PCT/US2009/051307 filed Jul. 21, 2009,which claims the benefit under 35 USC §119(e) to U.S. application Ser.No. 61/083,494 filed Jul. 24, 2008, now expired. The disclosure of eachof the prior applications is considered part of and is incorporated byreference in the disclosure of this application.

GRANT INFORMATION

This invention was made with government support under Grant No. GM20501awarded by the National Institutes of Health. The government has certainrights in the invention

BACKGROUND OF THE DISCLOSURE

1. Field of the Disclosure

The present disclosure relates generally to compounds useful for theinhibition of human secreted phospholipase A₂. More specifically, thedisclosure relates to the use of certain amides derived from non-naturalamino acids as inhibitors.

2. Background Information

The phospholipase A₂ (PLA₂) superfamily of enzymes consists of a broadrange of enzymes defined by their ability to catalyze the hydrolysis ofthe ester bond at the sn-2 position of phospholipids, yielding freefatty acids, including arachidonic acid, and lysophospholipids. Thecharacterization and classification of PLA₂ enzymes as well as theirrole in pathophysiological conditions are known. PLA₂ enzymes have beensystematically classified into 15 groups and subgroups on the basis oftheir nucleotide and amino acid sequence. According to a broaderclassification the PLA₂ classes have been historically classified intothree types: secretory (sPLA₂), cytosolic Ca²⁺-dependent (cPLA₂) andcytosolic Ca²⁺-independent (iPLA₂).

The Group IVA cPLA₂ (GIVA cPLA₂) is a particularly attractive target fordrug development, since it is the rate-limiting provider of arachidonicacid and lysophospholipids that can be converted into prostaglandins,leukotrienes and PAF, respectively. Various studies on gene-targetedmice that lack GIVA cPLA₂ showed that prostaglandins and leukotrieneproduction was reduced by approximately 90%, confirming the primacy ofGIVA cPLA₂ in lipid mediator production. Recently, it was demonstratedthat GIVA cPLA₂ plays an important role in the pathogenesis ofautoimmune encephalomyelitis (which models multiple sclerosis), and thatcytosolic phospholipase A₂-deficient mice are resistant to experimentalautoimmune encephalomyelitis.

The role of the other intracellular PLA₂, calcium-independent PLA₂ (GVIAiPLA₂), in the inflammatory process is still unclear, and it has notbeen a target for the development of novel medicines. This enzymeappears to be the primary PLA₂ for basal metabolic functions within thecell.

It has been shown that in macrophages and other cells, GIVA cPLA₂ andsecretory phospholipase A₂ work together to release arachidonic acid.Several experiments suggest that GV sPLA₂ has a role in amplifying theaction of GIVA cPLA₂ in supplying arachidonic acid for eicosanoidproduction. In addition, GV sPLA₂ has functions independent of itsability to provide arachidonic acid that include regulation ofphagocytosis and foam cell formation, suggesting a potential role ininflammatory processes such as atherosclerosis.

Amide phospholipids analogues of substrates can inhibit the activity ofsecreted PLA₂ (e.g., compound 1 shown in FIG. 1). Compound 2 (also shownin FIG. 1) is also as a potent inhibitor of sPLA₂. Non-phospholipidamide compounds based on non-natural amino acids, such as compounds 3and 4 (FIG. 1) can inhibit the activity of pancreatic and non-pancreaticGI and GII sPLA₂, and compound 4 has been reported to protect rat smallintestine from I/R injury and TNBS-induced colitis.

The selective inhibition of the various PLA₂ classes is very importantto understand their specific roles in cells and in vivo and developtherapeutic strategies accordingly. Accordingly, a variety of amidesbased on non-natural amino acids have been synthesized and theirspecific inhibitory activity on three human PLA₂ classes: GIVA cPLA₂,GVIA iPLA₂, and GV sPLA₂ demonstrated as described below in the presentapplication.

SUMMARY OF THE DISCLOSURE

The selective inhibition of the various PLA₂ classes is very importantto understand their specific roles According to embodiments of thepresent disclosure, there are provided compounds having the generalstructure A or pharmaceutically acceptable salts thereof are provided:

wherein R¹ is H, F, NH₂, or COOH; R² is, H, linear saturated orunsaturated alkyl, alkenyl, or alkynyl; each of R³ and R⁴ isindependently H, linear saturated or unsaturated alkyl, alkenyl,alkynyl, phenyl, or substituted phenyl; R⁵ is H, or (C₁-C₆)alkyl, suchas methyl or ethyl, X is aryl or substituted aryl, such as phenyl orsubstituted phenyl; and Y is O or S.

Pharmaceutical compositions of inhibitory compounds of the disclosurefor use in treating inflammatory conditions for which inhibition ofcPLA2, iPLA2 and/or sPLA2 is beneficial are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates several known inhibitors of phospholipases A₂.

FIG. 2 illustrates inhibition curve for an amide of the presentdisclosure in a mixed-micelle assay with human GV sPLA₂.

FIG. 3 illustrates the assessment of locomotor recovery after spinalcord injury (SCI) and administration of compound 9.

FIG. 4 illustrates the quantification of tissue and myelin sparing inmice treated with compound 9.

FIG. 5 illustrates the quantification of serotonergic fiber sparing andneuronal survival at 28 after SCI in mice treated with compound 9.

DETAILED DESCRIPTION OF THE DISCLOSURE

The following non-limiting definitions are provided for ease ofreference, and are intended to illustrate, rather than limit, the scopeof this disclosure.

The term “alkyl” refers to either substituted or unsubstituted C₁-C₁₀straight chain saturated aliphatic hydrocarbon groups, substituted andunsubstituted C₂-C₁₀ straight chain unsaturated aliphatic hydrocarbongroups, substituted and unsubstituted C₄-C₁₀ branched saturatedaliphatic hydrocarbon groups, substituted and unsubstituted C₄-C₁₀branched unsaturated aliphatic hydrocarbon groups, substituted andunsubstituted C₃-C₈ cyclic saturated aliphatic hydrocarbon groups,substituted and unsubstituted C₅-C₈ cyclic unsaturated aliphatichydrocarbon groups having the specified number of carbon atoms. Forexample, the definition of “alkyl” shall include but is not limited to:methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, ethenyl, propenyl, butenyl, penentyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, undecenyl, isopropyl, isobutyl, tert-butyl,sec-butyl, isopentyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl,cycloheptenyl, cyclooctenyl, methylcyclopropyl, ethylcyclohexenyl,butenylcyclopentyl, adamantyl, norbornyl and the like.

Alkyl substituents are independently selected from halogen, —OH, —SH,—NH₂, —CN, —NO₂, ═O, ═CH₂, trihalomethyl, carbamoyl, arylC₀₋₁₀alkyl,heteroarylC₀₋₁₀alkyl, C₁₋₁₀alkyl-oxy, arylC₀₋₁₀alkyloxy, C₁₋₁₀alkylthio,arylC₁₋₁₀alkylthio, C₁₋₁₀alkylamino, arylC₀₋₁₀alkyl-amino,N-aryl-N—C₀₋₁₀alkylamino, C₁₋₁₀alkylcarbonyl, arylC₀₋₁₀alkylcarbonyl,C₁₋₁₀alkyl-carboxy, arylC₀₋₁₀alkylcarboxy, C₁₋₁₀alkylcarbonylamino,arylC₀₋₁₀alkyl-carbonylamino, tetrahydrofuryl, morpholinyl, piperazinyl,hydroxypyronyl, C₀₋₁₀alkylCOOR_(a) and C₀₋₁₀alkyl-CONR_(b)R_(c), whereinR_(a), R_(b) and R_(c) are each independently selected from hydrogen,alkyl, and aryl, or R_(a) is as describe above, and R_(b) and R_(c) aretaken together with the nitrogen to which they are attached to form asaturated cyclic or unsaturated cyclic system containing 3 to 8 carbonatoms, optionally with at least one substituent.

The term “aryl” refers to an unsubstituted, mono-, di- or trisubstitutedmonocyclic, polycyclic, biaryl aromatic groups covalently attached atany ring position capable of forming a stable covalent bond, certainpreferred points of attachment being apparent to those skilled in theart (e.g., 3-phenyl, 4-naphtyl and the like). The aryl substituents areeach independently selected from halogen, —OH, —SH, —CN, —NO₂,trihalomethyl, hydroxypyronyl, C₁₋₁₀alkyl, arylC₀₋₁₀alkyl,C₀₋₁₀alkyloxyC₀₋₁₀alkyl, arylC₀₋₁₀alkyloxy-C₀₋₁₀alkyl,C₀₋₁₀alkylthioC₀₋₁₀alkyl, arylC₀₋₁₀alkylthioC₀₋₁₀alkyl,C₀₋₁₀alkylaminoC₀₋₁₀alkyl, arylC₀₋₁₀alkylaminoC₀₋₁₀alkyl,N-aryl-N—C₀₋₁₀alkylaminoC₀₋₁₀alkyl, C₁₋₁₀alkylcarbonyl-C₀₋₁₀alkyl,arylC₀₋₁₀alkylcarbonylC₀₋₁₀alkyl, C₁₋₁₀alkylcarboxyC₀₋₁₀alkyl,arylC₀₋₁₀alkyl-carboxyC₀₋₁₀alkyl, C₁₋₁₀alkylcarbonylaminoC₀₋₁₀alkyl,arylC₀₋₁₀alkylcarbonylaminoC₀₋₁₀alkyl, alkylCOOR_(a), and—C₀₋₁₀alkylCONR_(b)R_(c), wherein R_(a), R_(b) and R_(c) are eachindependently selected from hydrogen, alkyl, and aryl; or R_(a) is asdescribed above, and R_(b) and R_(c) are taken together with thenitrogen to which they are attached to form a saturated cyclic orunsaturated cyclic system containing 3 to 8 carbon atoms, optionallywith at least one substituent.

The definition of “aryl” includes, but is not limited to, such specificgroups as phenyl, biphenyl, naphthyl, dihydronaphthyl,tetrahydronaphthyl, indenyl, indanyl, azulenyl, anthryl, phenanthryl,fluorenyl, pyrenyl and the like.

The terms “halogen”, “halide” or “halo” refer to fluorine, chlorine,bromine, and iodine.

The abbreviation “BOC” refers to tert-butoxycarbonyl moiety having thestructure —C(O)—O—C(CH₃)₃.

The abbreviation “TEMPO” refers to teramethylpiperidin-1-oxyl moietyhaving the structure:

The term “effective amount” of a compound refers a non-toxic butsufficient amount of the compound that provides a desired effect. Thisamount may vary from subject to subject, depending on the species, age,and physical condition of the subject, the severity of the disease thatis being treated, the particular compound used, its mode ofadministration, and the like. Therefore, it is difficult to generalizean exact “effective amount,” yet, a suitable effective amount may bedetermined by one of ordinary skill in the art.

The term “pharmaceutically acceptable” refers to a compound, additive orcomposition that is not biologically or otherwise undesirable. Forexample, the additive or composition may be administered to a subjectalong with a compound of the disclosure without causing any undesirablebiological effects or interacting in an undesirable manner with any ofthe other components of the pharmaceutical composition in which it iscontained.

The term “pharmaceutically acceptable salts” includes hydrochloric salt,hydrobromic salt, hydroiodic salt, hydrofluoric salt, sulfuric salt,citric salt, maleic salt, acetic salt, lactic salt, nicotinic salt,succinic salt, oxalic salt, phosphoric salt, malonic salt, salicylicsalt, phenylacetic salt, stearic salt, pyridine salt, ammonium salt,piperazine salt, diethylamine salt, nicotinamide salt, formic salt, ureasalt, sodium salt, potassium salt, calcium salt, magnesium salt, zincsalt, lithium salt, cinnamic salt, methylamino salt, methanesulfonicsalt, picric salt, tartaric salt, triethylamino salt, dimethylaminosalt, tris(hydroxymethyl)aminomethane salt and the like. Additionalpharmaceutically acceptable salts are known to those of skill in theart.

As used herein, the term “patient” refers to organisms to be treated bythe methods of the present disclosure. Such organisms include, but arenot limited to, humans. In the context of the disclosure, the term“subject” generally refers to an individual who will receive or who hasreceived treatment for the treatment of a disease, disorder orpathology.

According to embodiments of the present disclosure, there are providedcompounds having the general structure A or pharmaceutically acceptablesalts thereof:

wherein R¹ is H, F, NH₂, or COOH; R² is, H, or a linear saturated orunsaturated alkyl, alkenyl, or alkynyl; each of R³ and R⁴ is H, a linearsaturated or unsaturated alkyl, alkenyl, or alkynyl, phenyl, or asubstituted phenyl; R⁵ is H, methyl, or ethyl, X is an aryl or asubstituted aryl, such as phenyl or a substituted phenyl; and Y is O orS.

One specific example of a useful compound encompassed by the genius ofthe general structure A is a compound where each of R¹, R³, R⁴, and R⁵is H, R² is n-butyl, X is phenyl, and Y is O (i.e.(R)-4-(7-phenylheptanamido)octanoic acid, or compound 9 shown below):

Various synthetic schemes can be designed for manufacturing the productshaving the structure A, including the specific compound 9. To exemplify,but not limit, the present disclosure, in one embodiment, the reactionscheme I shown below can be employed for making such compounds. Ifdesired, other synthetic processes can be designed by those havingordinary skill in the art.

More detailed discussion of the conditions and the results of thesynthesis shown by the reaction scheme I is provided below, in the“Examples” portion of the present application.

The following examples are intended to further illustrate but not limitthe scope of the disclosure. Standard abbreviations (e.g., “ml” formilliliters, and “min.” for minutes) are used.

EXAMPLES Example 1 Synthesis of (R)-4-(7-phenylheptanamido)octanoic acid

The synthesis of a γ-norleucine-based amide containing the7-phenylheptanoyl chain was conducted as shown by the reaction scheme Ishown above. Starting compound 5 (i.e., Boc-D-norleucinol) preparedaccording to the previously known techniques, was oxidized to aldehyde(step (a) on the reaction scheme I) using NaOCl, TEMPO, NaBr, andNaHCO₃. A 3:3:0.5 mixture of athyl acetate, toluene and water was usedas a solvent system. The reaction of oxidation was carried out at atemperature of about −5° C. As a step (b) shown by the reaction schemeI, methyl(phosphoranylidene)acetate Ph₃P═CHCOOMe or, alternatively,ethyl(phosphoranylidene)acetate Ph₃P═CHCOOEt in tetrahydrofuran wasadded and the system was brought to produce either compounds 6b orcompound 6a, respectively, as shown on the reaction scheme I.

After hydrogenation and removal of Boc group, using H₂, 10% Pd/C, andmethanol (step (c) shown by the reaction scheme I), coupling with7-phenylheptanoic acid gave amides 8a,b. The process of coupling wasconducted using 4N hydrochloric acid in ethyl ether (step (d) shown bythe reaction scheme I), and Ph(CH₂)₆COOH (i.e., 7-phenylheptanoic acid),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (i.e., WSCI), butylalcohol, triethylamine, and dichloromethane (step (e) shown by thereaction scheme I).

The title compound 9 was then obtained after saponification of either 8aor 8b the final step (f) shown by the reaction scheme I). 1N NaOH indioxane was used for saponification. The of the enantiomer of the titlecompound 9 was synthesized following similar reactions.

Example 2 In Vitro Inhibition of GIVA cPLA₂, GVIA iPLA₂ and GV sPLA2

Compound 9 based on (R)-γ-norleucine is a potent and selective GV sPLA₂inhibitor, not affecting at all the activities of GIVA cPLA₂ and GVIAiPLA₂. The dose-response curve for the inhibition of GV sPLA₂ bycompound 9 is shown in FIG. 2, and a non-linear regression (hyperbolic)led to the calculation of X_(I)(50) value of 0.003±0.0004. Theconfiguration of the amino acid is important, since its enantiomer basedon (S)-γ-norleucine was inactive for all the three PLA₂ enzymes.

The amides were tested for inhibition of human GIVA cPLA₂, GVIA iPLA₂and GV sPLA₂ using previously described mixed micelle-based assays. Theresulting degrees of inhibition are presented as either percentinhibition or X_(I)(50) values. Initially, the percent of inhibition foreach PLA₂ enzyme at 0.091 mole fraction of each inhibitor wasdetermined, and, X_(I)(50) values were estimated for compounds thatdisplayed greater than 90% inhibition. The X_(I)(50) is the molefraction of the inhibitor in the total substrate interface required toinhibit the enzyme by 50%. Compound 9 did not inhibit GIVA cPLA₂ andGVIA iPLA₂ at 0.091 mole fraction.

Example 3 Effects of Compound 9 in Spinal Cord Injury (SCI)

FIG. 3 illustrates the assessment of locomotor recovery after SCI. (A)Administration of compound 9 started 1 hour after SCI improved locomotorfunction assessed using the 9-point Basso Mouse Scale (BMS) as comparedwith the vehicle treated mice. Post-hoc analysis revealed significantdifferences in BMS score starting at day 5 dpi and remainingsignificantly enhanced during the duration of the follow up. (B) Finelocomotor skills assessed by the BMS subscores also showed a significantimprovement in mice treated with compound 9 from day 10 to 28 after SCI.*p<0.05.

FIG. 4 illustrates the quantification of tissue and myelin sparingassessed by staining for GFAP and luxol fast blue, respectively, at 28days after SCI. (A) Mice treated with compound 9 displayed a significantreduction tissue loss in tissue sections at 100 μm rostral and caudal tothe lesion epicenter. (B) Compound 9 treatment also led to significantmyelin sparing at the epicenter and areas rostral and caudal to it.

FIG. 5 illustrates the quantification of serotonergic fiber sparing andneuronal survival at 28 after SCI. (A) Mice treated with compound 9displayed significantly greater serotonergic sparing in the ventralhorns 1 mm caudal to the lesion epicenter. (B) Compound 9 also promotedgreater neuron survival in tissue section at 500 μm rostral and caudalto the lesion epicenter.

EXAMPLE 4. Inhibition of PLA₂ by Synthetic Inhibitors^(a) Inhibition (%at 0.091 mol fraction) cPLA₂ iPLA₂ sPLA₂ Inhibitor Structure GIVA GVIAGIIA/GV* AX115¹

62 ± 1 45 ± 13 52 ± 4 FKGK11²

N.D. >95 X_(I)(50) = 0.0096 ± 0.0008 28 ± 1 Compound 9¹

N.D. N.D. >95 X_(I)(50) = 0.003 ± 0.0004 ^(a)Average percent inhibitionand standard error (n = 3) reported for each compound at 0.091 molefraction. X_(I)(50) values determined for inhibitors with greater than95% inhibition. N.D. signifies compounds with less than 25% inhibition(or no detectable inhibition). References: ^(1.)“STRUCTURE-ACTIVITYRELATIONSHIPS OF NATURAL AND NON-NATURAL AMINO ACID-BASED AMIDE AND2-OXOAMIDE INHIBITORS OF HUMAN PHOSPHOLIPASE A₂ ENZYMES” G.Antonopoulou, E. Barbayianni, V. Magrioti, N. Cotton, D. Stephens, V.Constantinou-Kokotou, E. A. Dennis, and G. Kokotos Bioorg. Med. Chem.2008, 16, 10257-10269. ^(2.)“SYNTHESIS OF POLYFLUORO KETONES FORSELECTIVE INHIBITION OF HUMAN PHOSPHOLIPASE A₂ ENZYMES” C. Baskakis, V.Magrioti, N. Cotton, D. Stephens, V. Constantinou-Kokotou, E. A. Dennisand G. Kokotos J. Med. Chem. 2008, 51, 8027-8037.

The disclosure has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the disclosure. This includes thegeneric description of the disclosure with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the disclosure are described in terms of Markushgroups, those skilled in the art will recognize that the disclosure isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group. Further, the references appended heretoare all incorporated herein by this reference.

What is claimed is:
 1. A compound having the structure A, orpharmaceutically acceptable salts thereof:

wherein: R¹ is H, F, NH₂, or COOH; R² is H, linear saturated orunsaturated unsubstituted (C₂-C₁₀)alkyl, alkenyl, or alkynyl; each of R³and R⁴ is independently H, linear saturated or unsaturated alkyl,alkenyl, alkynyl, phenyl, or substituted phenyl; R⁵ is H or(C₁-C₆)alkyl; X is aryl or substituted aryl; and Y is O or S.
 2. Thecompound of claim 1, wherein R⁵ is H, methyl, or ethyl; and X is phenylor substituted phenyl.
 3. The compound of claim 1, wherein the compoundhas the formula 9:


4. A method of inhibiting a phoshpolipase A₂, comprising contacting thephoshpolipase A₂ with a compound having the structure A, orpharmaceutically acceptable salts thereof:

wherein: R¹ is H, F, NH₂, or COOH; R² is H, linear saturated orunsaturated unsubstituted (C₂-C₁₀)alkyl, alkenyl, or alkynyl; each of R³and R⁴ is independently H, linear saturated or unsaturated alkyl,alkenyl, alkynyl, phenyl, or substituted phenyl; R⁵ is H or(C₁-C₆)alkyl; X is aryl or substituted aryl; and Y is O or S.
 5. Themethod of claim 4, wherein R⁵ is H, methyl, or ethyl; and X is phenyl orsubstituted phenyl.
 6. The method of claim 4, wherein the compound hasthe formula 9: